Coccidiosis vaccines

ABSTRACT

Attenuated E. necatrix suitable for use in the production of a live vaccine is produced by passaging pathogenic E. necatrix in embryonated eggs for a sufficient number of passages i.e. from about 20 up to about 50 or more egg passages. The live egg-attenuated E. necatrix of the invention may be formulated in vaccines, e.g. feed or drinking water vaccines, for prevention and control of coccidiosis in poultry, usually with other strains of Eimeria (i.e. E. acervulina, E. brunetti, E. maxima, E. mivali, E. praecose and E. tenella) as live pathogenic strains thereof or preferably attenuated non-pathogenic lines thereof. In a preferred embodiment the egg-attenuated E. necatrix of the invention is formulated in a vaccine comprising attenuated, precocious E. acervulina.

This invention relates to coccidiosis vaccines, and in particular tovaccines for preventing and combatting coccidiosis in poultry.

Coccidiosis is a disease of animals and birds caused by protozoalparasites called coccidia. In many cases, under natural environmentalconditions, this disease is relatively benign; though with domesticatedanimals infections can be severe especially in poultry where the use ofintensive rearing conditions favours the reproduction of the parasites.Coccidiosis of poultry is caused by coccidia belonging to the genusEimeria which invade the tissue of the intestinal tract where somespecies of the parasite may cause haemorrhage, all species may causeweight loss and in the case of severe infection most may cause death.Thus the prevention and control of this disease is of great commercialimportance in the chicken and egg production industries.

Anticoccidial drugs are widely used to prevent and combat the disease ofcoccidiosis in poultry, the drugs normally being given to the birdstogether with their feed. The use of anticoccidial drugs is notaltogether satisfactory, however, in view of expense and alsoeffectiveness which is limited by emergence of drug resistant strains ofcoccidia requiring a continued search for new effect anti-coccidialagents and the attendant expense of meeting national safety and efficacyregulations.

Coccidiosis of chickens is caused by seven main species of Eimeriaparasites: E. acervulina, E. brunetti, E. maxima, E. mivati, E. praecox,E. necatrix and E. tenella; the coccidia undergoing and completing acomplex life cycle in the tissues of the intestinal tract of thechicken. The first invasive forms of the parasite are sporozoites whichare released from sporocysts derived from sporulated oocysts, thecoccidia then passing through a number of asexual multiplicative phases(schizogony) having merozoite invasive forms and culminating in a sexualreproductive phase (gametogony) giving rise to oocysts which are passedfrom the bird with faecal material and which, on becoming infective(sporulated), act as a source of further infection.

In view of the inherent disadvantages of anticoccidial drugs mentionedpreviously attempts have been made to develop vaccines to protectpoultry against coccidial infection. Hitherto, these vaccines havegenerally been based upon low doses of live, pathogenic organisms, andthus are not wholly desirable as it is possible that the vaccine itselfcan give rise to field cases of coccidiosis. More recently, some speciesof Eimeria parasites, namely E. mivati and E. tenella, have beenattenuated in embryonated eggs, and thus there now exists a limitedpossibility of preparing a vaccine from live, attenuated, non-pathogenicorganisms. Unfortunately, however, immunity is species specific and theattenuation of all Eimeria species is necessary for production of afully effective attenuated coccidiosis vaccine. Moreover, attempts toattenuate other species of Eimeria, including E. maxima, E. brunetti andespecially the highly pathogenic species E. necatrix, have provedunsuccessful, E. necatrix, appearing to be capable of completing thesexual reproductive stages of its life cycle only in the natural host,the chicken. Growth of E. necatrix through its whole life cycle inembryonated eggs was reported some time ago (Shibalova, ActaProtozoologica, Warszawa, 1972, Volume IX, fasc. 19, pages 299-303), butsubsequent attempts to repeat this work have proved completelyunsuccessful (P. L. Long, "The growth of Eimeria in cultured cells andin chicken embryos: a review", Proceedings of the Symposium on Coccidiaand Related Organisms, Ontario 1974 page 63), and it had not beengenerally accepted that E. necatrix can be propagated through itscomplete life cycle in embryonated eggs.

It has now been found, contrary to expectation, that E. necatrix can bepropagated throughout its complete life cycle in embryonated eggs, andfurther that repeated passaging of this organism in embryonated eggsleads to attenuation of the parasite.

Accordingly the present invention comprises a process in whichpathogenic E. necatrix is passaged in embryonated eggs for a sufficientnumber of passages to produce an attenuated strain of E. necatrixsuitable for use in the production of a live, attenuated vaccine.

Thus, the invention also includes egg attenuated strains of E. necatrixsuitable for use in live attenuated vaccines.

Furthermore the invention includes vaccines, processes for theproduction of vaccines and methods of preventing and controllingcoccidiosis in poultry using attenuated strains of E. necatrix accordingto the invention.

Eggs from any suitable breed of chicken may be used for attenuation ofE. necatrix according to the invention, and in particular attenuation iscarried out in the chorioallantoic membrane (CAM) of embryonated eggs.It has been found that eggs of some breeds of chickens appear to givebetter yields of parasite oocysts than eggs of other breeds. Thus, forinstance, embryonated Rhode Island Red (RIR) eggs appear to giveincreased yields of oocysts as compared with Brown Leghorn (BrL) eggs.

Generally also, any suitable in ovo culture conditions may be employedduring attenuation of E. necatrix. Advantageously, however, it has beenfound that relatively elevated temperatures, e.g. temperatures of about41° C., e.g. 39°-43° C., are desirable for growth of E. necatrix inembryonated eggs. Furthermore, it has been found that regular turning ofeggs, e.g. a minimum of at least once or twice a day, is desirableduring propagation in the CAM, and for example, an automatic incubationfacility which automatically turns the eggs about 48 times during thecourse of one day has been found to give highly successful results.

Embryonated eggs may be inoculated with sporozoites of E. necatrix andoocysts subsequently recovered from the eggs, usually from the urate andother debris present in the allantoic fluid. Initially it may bedesirable to use relatively high doses of sporozoites, usually about1×10⁵ sporozoites or more per egg, e.g. from 1×10⁵ up to 1×10⁷sporozoites per egg, to establish an egg-adapted strain, thoughsubsequently, e.g. after about 13-14 passages, lower doses, e.g. about5×10⁴ sporozoites or less per egg, are generally required. Also it maybe desirable, particularly during the initial stages of passaging e.g.for about the first 6-10 egg passages, to carry out alternate passagesin eggs and chickens so as to obtain a reasonable yield of oocysts.Harvesting of oocysts may be carried out after a suitable period ofincubation for each passage, usually at least 6 or 7 days afterinoculation, though preferably after a longer period, e.g. about 7-8days. Relatively short incubation periods, e.g. 6 or 7 days or even aslittle as 5 days in some cases, may be used when selection forprecocious development is imposed on the parasite during passaging.During the earlier passages, however, longer periods of incubation, e.g.about 8 days, are desirable to obtain satisfactory yields of oocysts,and such longer incubation periods may also be used during later stagesof passaging to obtain enhanced oocyst yields.

Typically passaging is continued for a sufficient number of passages toproduce a non-pathogenic strain of E. necatrix of suitableimmunogenicity and stability for use in a live attenuated vaccine. Inthis regard, E. necatrix appears to behave anomalously on passaging whencompared with other species of Eimeria, e.g. E. tenella, which have beenpreviously attenuated by passaging in eggs. It has been found that inparallel with loss of pathogenicity on passaging immunogenicity alsodecreased, and furthermore that stability has not yet become agenetically stable trait at the stage at which useful immunogenicitystill remains. Thus an attenuated strain of E. necatrix according to theinvention represents an acceptable compromise between the opposingtendencies of decreasing immunogenicity as against increasing stability,coupled with non-pathogenicity.

It has been found in accordance with the present invention that fromabout 20 up to about 50 passages or more are required to produce astrain of E. necatrix suitable for use in a live attenuated vaccine. Forinstance, up to about 50 or 60 egg passages may be used when selectionpressures such as selection for precocious development are relaxedduring passaging; though under such relaxed selection conditions, fromabout 30 up to about 50 passages are normally required, preferably fromabout 35 to about 39 passages. Alternatively when selection pressures,such as selection for precocious development, are imposed, asspecifically described hereinafter, normally from about 20 up to about40 passages, preferably from about 25 up to about 35 passages, orespecially from about 28 up to about 33 passages are used.

The attenuated strains of the invention are characteristically alteredwith respect to the parent pathogenic strain by the passaging procedureand may be distinguished therefrom in terms of pathogenicity andadaptation to growth in eggs.

Thus attenuated strains of the invention typically do not cause deathwhen administered to susceptible chickens, e.g. at doses of about 5×10⁴of fresh oocysts per chicken. Preferably also, the attenuated strainscause only relatively minor changes to the gross appearance of theintestine on infection, and especially cause no actual weight loss(weight loss being determined on a group basis) when administered tochickens at doses of about 5×10⁴ of fresh oocysts per bird.

Additionally, the attenuated strains are typically of stability suchthat they are of reduced pathogenicity when compared with the parentpathogenic strain, after they have undergone at least 5, e.g. 5 or 6, orespecially 10, consecutive chicken passages after attenuation and beforeadministration to the susceptible chickens. Thus, for example anattenuated strain, attenuated by 29 passages in eggs followed by 6consecutive passages in chickens, causes less than 50%, e.g. 30%,mortality in chickens at a dose of 5×10⁴ oocysts per chicken, comparedwith 100% mortality caused by the same dose of the pathogenic parentstrain.

Thus, it will be appreciated that strains of E. necatrix attenuatedaccording to the invention even after subsequent passaging in chickensare more advantageous, from the point of view of pathogenicity, forvaccine use than the parent pathogenic strains. Typically also theattenuated strains provide reasonable protection against subsequentchallenge with virulent E. necatrix. Thus, attenuated strains usuallyprovide at least 85% and preferably at least 95% protection againstchallenge with virulent E. necatrix, for example, as reckoned by aprocedure in which immunised and non-immunised birds are given a smallchallenge of virulent organisms and the resultant output of oocysts inthe immunised and non-immunised birds are compared.

Generally also attenuated strains according to the invention typicallyreproduce in chickens less readily, though grow in eggs more easily,than the parent pathogenic strains. Thus, for example, a 5×10³ dose ofoocysts of the parent strain produces a yield of about 25×10⁶ oocysts inchickens whereas a 25×10³ dose of an attenuated strain after 29 eggpassages produces only about 1×10⁶ oocysts, and after 36 egg passagesproduces only about 0.4×10⁶ oocysts. Conversely, as regards growth ineggs, for example, a 10⁵ dose of sporozoites of the parent pathogenicstrain per egg gave a yield of only 3×10³ oocysts after 8 daysincubation; whereas a 2.2×10⁴ dose of sporozoites per egg of a strainattenuated by 29 egg passages gave a yield of 560×10³ oocysts after only6 days incubation.

Attenuated non-pathogenic strains of E. necatrix according to theinvention may be formulated as desired into vaccines for prevention andcontrol of coccidiosis in poultry. Attenuated E. necatrix, which may bein the form of sporocysts, though is usually in the form of oocysts, maybe formulated into vaccines with other strains of Eimeria including livepathogenic strains of Eimeria such as E. maxima, E. acervulina and E.brunetti, usually as low doses thereof, and/or preferably otherattenuated non-pathogenic strains of Eimeria such as E. tenella and E.mivati. Such combined coccidiosis vaccines preferably contain, inaddition to attenuated E. necatrix, strains of all the major coccidia ofpoultry, i.e. E. acervulina, E. brunetti, E. maxima, E. mivati and E.tenella, preferably as attenuated strains thereof, and may also containstrains of E. praecox to provide a fully effective vaccine for useagainst coccidiosis of poultry.

In preferred embodiments vaccines according to the present inventioncomprise, in addition to egg attenuated E. necatrix, egg attenuated E.tenella and/or egg attenuated E. mivati, and/or E. acervulina attenuatedby selection for precocious development in the chicken. In addition oralternatively E. tenella, and possibly also E. mivati, may be attenuatedby selection for precocious development in the chicken. Vaccinesaccording to the invention may also comprise low doses of pathogenic E.maxima, this species being highly immunogenic and therefore requiringonly a low dose for effective protection.

Vaccines according to the invention may also comprise antigenic materialfrom other species of organisms besides Eimeria, such as viruses.

The vaccines of the invention may be in any suitable form foradministration to poultry including those forms in which coccidiosisvaccines have been provided in the past, e.g. COCCIVAC. Vaccine istypically in the form of a suspension of oocysts or sporocysts, normallya suspension in a sterile aqueous medium, which may contain suspendingagents such as gelatin. Oocysts or sporocysts may be pretreated prior tovaccine formulation; for instance, treated with an agent e.g.hypochlorite or "Chlorox", to render the oocysts more readily infectivee.g. for use with young birds. The vaccine may be administered to birdsby intravenous or intraperitoneal injection, but this is generally aninefficient ultilisation of the vaccine and vaccine is preferablyadministered by mouth. For instance, vaccine may be given individuallyby mouth in the form of graded doses of oocysts, e.g. up to about 5×10³oocysts of each strain per bird, though such individual vaccination isnormally only economically feasible for vaccination of layer,replacement and breeder birds. In preferred methods for vaccination ofbroiler chickens, oocysts are given to the chickens together with theirfeed, e.g. in bulk feed or in a feed concentrate alternative, ordrinking water. Feed and drinking water vaccination may be carried outby administering small doses of oocysts, e.g. 10-10³ oocysts of eachstrain per day, over an extended period of time, e.g. 1-5 weeks.Alternatively drinking water vaccination may be carried out by givingthe birds one or two larger doses of about 5×10³ oocysts e.g. from about5×10² up to about 5×10⁴ oocysts, of each strain per bird. Such feed anddrinking water vaccines may be made from concentrated stock vaccinepreparations, usually comprising aqueous suspensions of oocysts.

The present invention is concerned with the preparation of a "live"vaccine, and thus it will be appreciated that the attenuated strains orthe vaccine itself may be used as a seed material for production ofoocysts or sporocysts, preferably by propagation in eggs, for use invaccine. Thus the invention includes per se attenuated strains of E.necatrix produced by the method of the invention. Birds may bevaccinated at any suitable age, and are usually at least 3 days oldbefore first vaccination, though it may be possible to vaccinate asearly as one day old if sporozoites are used. When two doses of vaccineare used, the first is normally given when the birds are 3 days to aweek old and subsequently after a further 1-10 weeks dependent upon thetype of bird being vaccinated.

Generally also the birds are preferably maintained under conditionswhich permit them access to their litter allowing reinfection withoocysts derived from the vaccine, and thereby advantageously increasingthe level of immunological protection. For example, the litter may bedampened periodically to assist sporulation of the oocysts.

It will be appreciated from the foregoing that the present invention isprimarily concerned with processes for attenuation of pathogenic E.necatrix by passaging in embryonated eggs, advantageously to providelive attenuated E. necatrix vaccines for protection of poultry. In asfar, however, as the live attenuated strains of E. necatrix produced onpassaging are new organisms produced by human intervention from theparent pathogenic strain, the present invention also concerns new anduseful organisms. Thus, for better exemplification of the invention,samples of representative attenuated strains of E. necatrix have beendeposited with the Central Veterinary Laboratory, Weybridge, Surrey,England, a U.K. Government laboratory having extensive experience ofmaintaining cultures of poultry parasites, including coccidia. Culturesof strains of E. necatrix attenuated by 21, 32 and 48 egg passages weredeposited with the Central Veterinary Laboratory on the 21st August 1980and are identified as strains L/5B/G/21, L/5B/G/32 and L/5B/G/48respectively, and also as strains B3/6/R/21, B3/6/R/32 and B3/6/R/48respectively. These and other attenuated strains of E. necatrix of theinvention exhibit the typical characteristics of coccidia of species E.necatrix, though having pathogenicity and adaptation of growth in eggsaltered as herein described with respect to the parent pathogenicstrain.

The invention is further described by way of illustration only inExamples 1 and 2 which relate to the attenuation of E. necatrix inembryonated eggs and refer to the accompanying diagrams:

FIG. 1 which is a graph comparing body weight gains of chickens afterinfection with parent pathogenic organisms with those of chickensinfected with organisms attenuated by 20 chicken passages, and

FIG. 2 which is a similar graph comparing strains attenuated by 36 eggpassages, and 29 egg passages plus 6 consecutive chicken passages withthe fully pathogenic parent strain.

Example 3 is included to describe a method, as yet unpublished, forattenuating E. acervulina by selection for precocious development in thechicken and refers to FIG. 3 which is a graph comparing body weightgains of chickens after infection with the parent pathogenic strain ofE. acervulina (H) with those of chickens infected with the attenuated,precocious line of E. acervulina (HP).

EXAMPLE 1

The Houghton (H) strain of E. necatrix isolated at Houghton in December1956 and described by Horton-Smith and Long (Journal of ComparativePathology and Therapeutics (1959), 77, 315-325) was used as the startingmaterial in the production of an attenuated strain of E. necatrixaccording to the invention.

Sporozoites were obtained as described by Long (Journal of ComparativePathology (1972), 82, 439-445) and were inoculated into the allantoiccavity of eggs by the method of Long (Journal of Comparative Pathology(1972), 82, 429-437).

In a preliminary experiment, thirty 10-day-old embryonating BrownLeghorn (BrL) eggs given 1.3×10⁵ sporozoites of E. necatrix (H) wereexamined for oocysts 6, 7 and 8 days after inoculation. In one eggexamined on the sixth day no oocysts were found, but an average of2.5×10³ oocysts was recovered from each of 9 eggs on the seventh day. At190 h the urate of the remaining 20 eggs was examined and a total of0.39×10⁶ oocysts were recovered. These oocysts sporulated and weresubsequently inoculated into 3-week-old Light Sussex (LS) chickens toconfirm the identity of the parasites recovered from eggs as E.necatrix.

The embryonating eggs used in the above and subsequent experiments weremaintained at 37° C. for the first 10 days of incubation and afterinoculation at this time with sporozoites the temperature was raised to41° C. Similarly occysts were recovered from eggs 6, 7 or 8 days afterinoculation using the trypsin-digest method described by Long in thesecond of the above mentioned Journal of Comparative Pathologypublications.

Serial Passage

A dose response experiment in which eggs were inoculated with 0.5, 1, 2or 3×10⁵ sporozoites indicated that the optimum dose with regard toyield of oocysts was 1×10⁵. Yields were consistently poor, however, evenwith this dose and in order to establish an egg-adapted line it wasfound necessary initially to carry out alternate egg and chickenpassages, up to the first eight egg passages. Replacement of BrL eggs byRIR eggs, and turning of the eggs in the incubator, however, was foundto increase the number of oocysts recovered on the eighth day afterinoculation. In view of this improvement the inoculum of sporozoites wasreduced to about 6×10⁴ sporozoites per egg by the sixteenth passage.

Initially very few oocysts appeared in the urate by the seventh day andit was found necessary to continue incubation until after the eighth dayto obtain sufficient parasites for further passages. Oocysts recoveredat this time sporulated well and sporulation rates between 80 and 90%were usual.

Little mortality due to coccidiosis was observed throughout thepassages, although some deaths occurred between 4 and 6 days afterinoculation. These, and other data which summarise the development of E.necatrix during the first twenty passages in eggs, are given in Table 1below.

With the apparent step-wise improvement in yield of oocysts particularlybetween the eighth and fourteenth passage, it became possible thereafterto recover sufficient parasites 7, and even 6 days after inoculation andto impose upon the parasite a more severe selection for egg-adaptation.

                  TABLE 1                                                         ______________________________________                                        The reproduction of E. necatrix in eggs during serial passage                 Pas-          Number    Day after         Mor-                                age   Breed   sporozoites                                                                             inoculation                                                                            Oocysts  tal-                                Num-  of egg  given/egg when oocysts                                                                           recovered/                                                                             ity                                 ber   used    (× 10.sup.-3)                                                                     harvested                                                                              egg(×10.sup.-3)                                                                  (%)                                 ______________________________________                                        1.sub.0                                                                             BrL     100       8        3        0                                   2.sub.1                                                                             BrL     50        8        3        0                                   3.sub.2                                                                             BrL     100       8        2        7                                   4.sub.3                                                                             BrL     100       8        12       19                                  5.sub.3                                                                             BrL     50        8        3        0                                   6.sub.4                                                                             BrL     100       8        2        0                                   6.sub.4                                                                             BrL     150       8        10       15                                  6.sub.4                                                                             BrL     200       8        4        5                                   7.sub.6                                                                             BrL     110       8        4        5                                   7.sub.6                                                                             RIR     110       8        65       2                                   8.sub.7                                                                             RIR     90        8        40       1                                   8.sub.7                                                                             RIR     45        8        7        0                                   9.sub.7                                                                             RIR     90        8        56       1                                   10.sub.7                                                                            RIR     110       8        450      7                                   11.sub.7                                                                            RIR     100       8        340      0                                   12.sub.7                                                                            RIR     85        8        115      5                                   13.sub.7                                                                            RIR     100       8        910      2                                   14.sub.7                                                                            RIR     100       8        1,300    0                                   14.sub.7                                                                            RIR     50        8        730      0                                   14.sub.7                                                                            RIR     100       7        850      0                                   15.sub.7                                                                            RIR     100       7        750      1                                   16.sub.7                                                                            RIR     85        6 (140h) 10       2                                   17.sub.7                                                                            RIR     65        7        1,280    0                                   18.sub.7                                                                            RIR     55        6 (140h) 10       0                                   19.sub.7                                                                            RIR     53        7        1,330    0                                   20.sub.7                                                                            RIR     52        7        840      0                                   ______________________________________                                    

The numerical code used to indicate passage number in Table 1 comprisesa main number indicating the number of egg passages and a subscriptnumber indicating the number of chicken passages used, and is employedelsewhere in the description. Thus E. necatrix NA 20₇ indicates anegg-adapted line of E. necatrix which has undergone 20 passagesincluding initially 7 intermittent passages in chickens.

Effect of E. necatrix (H) and E. necatrix (NA 20₇) on the body weightgain of 3-weeks-old LS chickens and lesion scores Body weight gain

For this experiment five groups each consisting of three sub-groups often chickens were used. Group 1 was given 1×10⁴ oocysts of E. necatrix(NA 20₇); group 2 was given 1×10⁴ oocysts of E. necatrix (H); group 3was given 5×10⁴ oocysts of E. necatrix (NA 20₇) and group 4 was given5×10⁴ oocysts of E. necatrix (H). Group 5 was not infected. The birdswere weighed individually one day before infection (D-1), and 4, 6, 7,8, 11 and 14 days after infection. The body weights are shown in theaccompanying diagram FIG. 1, each value given being the mean determinedfrom observations on the 30 chickens (or survivors) in each group.Details of the deaths occurring in inoculated groups (1-4) are given inTable 2 below.

                  TABLE 2                                                         ______________________________________                                        Pathogenicity of E. necatrix (NA 20.sub.7) and E. necatrix (H):               Summary of mortality                                                                             Per-                                                                          cent-                                                      Parasite and       age     Days after infection on                            dose of oocysts                                                                         Number   mor-    which deaths occurred                              given     of birds tality  5   6   7   8   9   10  11                         ______________________________________                                        NA20 1 × 10.sup.4                                                                 30       0       0   0   0   0   0   0   0                          H   1 × 10.sup.4                                                                  30       3       0   1   0   0   0   0   0                          NA20 5 × 10.sup.4                                                                 30       0       0   0   0   0   0   0   0                          H   5 × 10.sup.4                                                                  30       40      2   5   3   2   0   0   1                          ______________________________________                                    

The non-passaged parent strain (E. necatrix (H)) was found to be highlypathogenic and caused loss in body weight beginning 4 days afterinoculation at both dose levels, the weight loss being greatest for thehighest dose level. One chicken given 1×10⁴ oocysts died, whilst 13(40%) died in the group given 5×10⁴ oocysts. In contrast no deathsoccurred in the groups inoculated with the egg-adapted line and, althughthe weight gain of infected chickens was depressed with respect to thecontrols, there was no weight loss. From the 6th day after inoculationthe growth of chickens within groups 1 and 2 was significantly different(P<0.001) from the growth of chickens in groups 3 and 4.

Lesion Scores

Twenty chickens, each given 5×10⁴ oocysts of E. necatrix (NA 20₇) or E.necatrix (H) were killed 6 days after inoculation and the intestinallesions graded according to an arbitary scale between 0 and 4.

Grade 0 indicated no gross lesions; grade 1 presence of small scatteredpetechiae and white spots visible from the serosal surface; grade 2,numerous petechiae on the serosal surface and some slight ballooning ofthe intestine; grade 3 for extensive haemorrhage in the lumen and thepresence of red or brown mucus, extensive petechiae on the serosalsurface, marked ballooning of the intestine and the absence of normalintestinal contents. Grade 4 was reserved for dead birds.

Mean values obtained for the two infections were 3.2 and 1.2 for E.necatrix (H) and E. necatrix (NA 20₇) respectively; 6 chickens given E.necatrix (H) died.

Immunogenicity of E. necatrix (NA 20₇) and E. necatrix (H)

Two groups of 20, 3-weeks-old LS chickens maintained in four sub-groupsof five, were given 1×10⁴ oocysts of either E. necatrix (NA 20₇) or E.necatrix (H). Twelve days later both groups, with a further group ofpreviously uninfected chickens, were challenged with 5×10² oocysts of E.necatrix (H). Oocyst production between 6 and 13 days after inoculationwas measured and the results are given below in Table 3.

                  TABLE 3                                                         ______________________________________                                        Cross-protection between E. necatrix (NA 207) and E. necatrix (H)                                 Total oocysts produced                                                                        Percent                                         Parasite used for                                                                           (× 10.sup.-6) per bird after                                                            cross-                                          primary inoculation                                                                         challenge inoculation                                                                         protec-                                   Group (Dose: 1 × 10.sup.4                                                                   with 5 × 10.sup.2 oocysts                                                               tion (c.f.                                No.   oocysts)      of E. necatrix (H)                                                                            Group 3)                                  ______________________________________                                        1     E. necatrix (NA 20.sub.7)                                                                   3.55            85.00                                     2     E. necatrix (H)                                                                             0.01            99.99                                     3     None          23.67                                                     ______________________________________                                    

Chickens given the parent strain (E. necatrix (H)) were almostcompletely protected against homologous challenge whereas those giventhe egg-adapted line showed 85% protection when compared with thenon-immunised group challenged with E. necatrix (H). Two further groupsof 10 chickens, similarly given a primary dose of 1×10⁴ oocysts of E.necatrix (NA 20₇) or E. necatrix (H) were challenged, together with anon-immunised group, with 5×10⁴ oocysts of E. necatrix (H). Thesechickens were killed after 6 days and the lesions graded. Mean lesionscores per chicken for the three groups were 2.35 (E. necatrix (NA20₇)), 0.05 (E. necatrix (H)) and 2.80 (non-immunised controls).

EXAMPLE 2

E. necatrix (NA 20₇) as produced in Example 1 was passaged further ineggs up to a total of 40 egg passages. Information concerning thedevelopment of E. necatrix during these further twenty passages is givenbelow in Table 4, being an extension of the information already givenfor the first twenty passages in Table 1, Example 1. Selection pressurefor precocious development was continued during these further passages,parasites having been collected at 5 or 6 days after inoculation afterthe 23rd passage. It will be appreciated, however, that considerablyhigher yields of oocysts can be obtained if selection pressures arerelaxed and oocysts recovered at a later stage after inoculation e.g. 7or 8 days, during these and previous passages.

                  TABLE 4                                                         ______________________________________                                        Reproduction of E. necatrix during serial passage in RIR eggs:                oocysts collected 5, 6 or 7 days after inoculation of sporozoites             Pass-                                                                              No. sporozoites                                                                           Day after inoculation                                                                        No. oocysts                                   age  given per egg                                                                             when oocysts were                                                                            recovered per                                 No.  (× 10.sup.-3)                                                                       recovered      egg (× 10.sup.-3)                       ______________________________________                                        21.sub.7                                                                           50          6              110                                           22.sub.7                                                                           33          6              210                                           23.sub.7                                                                           29          5 (125-127h)   1                                             24.sub.7                                                                            1          7              10                                            25.sub.7                                                                            4          6              140                                           26.sub.7                                                                           20          6              640                                           27.sub.7                                                                           26          6              60                                            28.sub.7                                                                           20          6              50                                            29.sub.7                                                                           22          6              560                                           30.sub.7                                                                           39          5 (125-127h)   20                                            31.sub.7                                                                           44          5 (123-125h)   5                                             32.sub.7                                                                           27          6              740                                           33.sub.7                                                                           23          5 (125-127h)   5                                             34.sub.7                                                                           20          6              1,000                                         35.sub.7                                                                           28          5 (125-127h)   20                                            36.sub.7                                                                           25          6              300                                           37.sub.7                                                                           25          5 (123.sub.125h)                                                                             30                                            38.sub.7                                                                           20          5 (123-125h)   10                                            39.sub.7                                                                           15          6              610                                           40.sub.7                                                                            8          6              980                                           ______________________________________                                    

The immunogenicity of the egg-adapted line was determined after a totalof 22 egg passages.

Immunogenicity of E. necatrix (NA 22₇) in chickens maintained in floorpen isolators

Four groups of 30, 3-week-old chickens were used. Group 1 was given1×10² oocysts of E. necatrix (NA 22₇); group 2 was given 1×10² oocystsof E. necatrix (H) and groups 3 and 4 were not inoculated with oocysts.Six weeks later (day 41) groups 1, 2 and 3 were challenged with 5×10⁴oocysts of E. necatrix (H). Group 4 was maintained as a non-immunised,non-challenged control group. The body weights of all chickens weremeasured at the time of primary infection (day 0), 41 days later--at thetime of challenge--and 7 days after the challenge infection (day 48).All chickens were killed at the latter time and the lesions graded.

Body weight gains of immunised chickens (groups 1 and 2) andnon-immunised control chickens in group 4 were not significantlydifferent 41 days after the primary infection as shown in Table 5 below.The body weight gain of group 3 was significantly different to that ofgroup 1 only (P<0.05).

                  TABLE 5                                                         ______________________________________                                        Body weight changes of chickens given E. necatrix and maintained              in litter pen isolators                                                       ______________________________________                                        Group Parasite used for                                                                          Initial    Weight gain number                              num-  immunisation body weight                                                                              day 0 to day 41                                 ber   (1 × 10.sup.2 oocysts)                                                               (g) (day 0)                                                                              (g)                                             ______________________________________                                        1     E. necatrix  197.7      864.5                                                 (NA 22)                                                                 2     E. necatrix (H)                                                                            197.7      814.1                                           3     None         197.7      785.0*                                          4     None         197.3      802.5                                           ______________________________________                                              Challenged with                                                         Group 5 × 10.sup.4 oocysts                                                                 Weight gain                                                                              Mean intestinal lesion                          num-  of E. necatrix (H)                                                                         day 41 to  score 7 days after                              ber   at day 41    day 48 (g) challenge                                       ______________________________________                                        1     YES          159.8      0                                               2     YES          158.3      0                                               3     YES          -19.2***   2.2                                             4     NO           164.7                                                      ______________________________________                                         ***Significantly different from Groups 1, 2 and 4 (P<0.001)?                  *Significantly different from Group 1 (P<0.05)?                          

Following challenge with 5×10⁴ oocysts of E. necatrix (H) none of theimmunised chickens died and their mean body weights (i.e groups 1 and 2)were not significantly different from that of group 4. In contrast, onechicken in group 3 died and the mean body weight was severely depressedwith respect to group 4 (P<0.001). No coccidial lesions were seen in anyof the chickens in groups 1 and 2 but a mean lesion score of 2.2 wasrecorded for group 3.

Stability of loss of pathogenicity of E. necatrix (NA 29₇)

A preliminary experiment was conducted using two groups of 13 and one of12, 3-weeks-old chickens. Each group was given 5×10⁴ oocysts and group 1received E. necatrix (H); group 2 received E. necatrix (NA 30₇) andgroup 3 received E. necatrix (NA 29₇ +1). (The reference NA 29₇ +1indicates that the organisms had been passaged 29 times in eggs followedby a single passage in chickens.) Six days after inoculation thechickens were killed and the intestinal lesions scored.

Mean lesion scores for the groups given E. necatrix (H), E. necatrix (NA30₇) and E. necatrix (NA 29₇ +1) were 2.62, 0 and 0.42 respectively, theoverall results being given below in Table 6. Although the loss ofpathogenicity of the egg-adapted line did not appear to be geneticallystable, it is clear that full virulence (i.e. to that of the parentstrain) was not restored. E. necatrix (NA 29₇ +1) was passaged inchickens a further 5 times and its pathogenicity was then morecritically evaluated against both the parent Houghton strain and theegg-adapted line.

                  TABLE 6                                                         ______________________________________                                        Lesion scores associated with infections of E. necatrix (H);                  E. necatrix (NA 30.sub.7) and E. necatrix (NA 29 + 1)                                      Number of chickens showing a                                                  lesion score of:-                                                Parasite given 0     1/2   1   11/2                                                                              2   21/2                                                                              3   31/2                                                                              4                          ______________________________________                                        E. necatrix (H)                                                                               0    0     0   1   3   1   6   1   0                          E. necatrix (NA 29 + 1)                                                                       6    3     4   0   0   0   0   0   0                          E. necatrix (NA 30)                                                                          13    0     0   0   0   0   0   0   0                          ______________________________________                                    

Pathogenicity of E. necatrix (H), E. necatrix (NA 36₇) and E. necatrix(NA 29₇ +6) in 3-week-old LS chickens Body weight gain

Five groups each consisting of 3 sub-groups of 10 chickens were used.Group 1 was given 5×10⁴ oocysts of E. necatrix (NA 36₇), group 2 wasgiven 2×10⁵ oocysts of E. necatrix (NA 36₇), group 3 was given 5×10⁴oocysts of E. necatrix (NA 29₇ +6) and group 4 was given 5×10⁴ oocystsof E. necatrix (H). Group 5 was not inoculated, the chickens wereweighed individually and assigned to the groups one day beforeinoculation (day -1) and then re-weighed 4, 6, 7, 8, 11 and 14 daysafter inoculation. The results obtained are given in the accompanyingdiagram FIG. 2, each value being the mean determined from observationson the 30 chickens (or survivors) in each group. No chickens givenoocysts of E. necatrix (NA 36₇) died, and throughout the duration of theexperiment there were no significant differences between the weightgains of groups 1 and 2 and the non-infected control group 5. (Theweight gains of groups 1 and 2 given 5×10⁴ and 2×10⁵ oocysts of E.necatrix (NA 36₇), respectively, were identical and thus results forgroup 1 alone are shown.)

Severe coccidiosis resulted from inoculation of 5×10⁴ oocysts of both E.necatrix (NA 29₇ +6) and E. necatrix (H) and the mortality rates were10% (3/30) and 60% (18/30) respectively. The weight gains of thesechickens were severely retarded with the greatest effect beingassociated with E. necatrix (H) infection.

Lesion Scores

Groups of 10, 3-weeks-old chickens were inoculated with 5×10⁴ oocysts ofeither E. necatrix (H), E. necatrix (NA 36₇) or E. necatrix (NA 29₇ +6)or 2×10⁵ oocysts of E. necatrix (NA 36₇). Six days later the chickenswere killed and the intestinal lesions scored. No lesions wereassociated with E. necatrix (NA 36₇) infection, whereas all chickensgiven E. necatrix (H) died (lesion score of 4) and those given E.necatrix (NA 29₇ +6) showed a mean lesion score of 3.15. The resultsobtained are given more fully in Table 7 below.

                  TABLE 7                                                         ______________________________________                                        Lesion scores associated with infections of E. necatrix (H)                   E. necatrix (NA 36.sub.7) and E. necatrix (NA 29.sub.7 + 6)                                  Numbers of chickens showing a lesion                           Parasite and dose                                                                            score of:-                                                     of oocysts given                                                                             0     1/2   1   11/2                                                                              2   21/2                                                                              3   31/2                                                                              4                          ______________________________________                                        E. necatrix (H)                                                                               0    0     0   0   0   0   0   0   10                         (5 × 10.sup.4)                                                          E. necatrix (NA 36.sub.7)                                                                    10    0     0   0   0   0   0   0    0                         (5 × 10.sup.4)                                                          E. necatrix (NA 36.sub.7)                                                                    10    0     0   0   0   0   0   0    0                         (2 × 10.sup.5)                                                          E. necatrix (NA 29.sub.7 + 6)                                                                 0    0     0   0   0   5   0   2    3                         (5 × 10.sup.4)                                                          ______________________________________                                    

Reproduction of E. necatrix (H), (NA 23₇), (NA 29₇), (NA 33₇) and (NA36₇) in chickens

To compare the reproductive potential of the parasites, groups of 15chickens (three sub-groups of 5) were given either 5×10³ oocysts of E.necatrix (H) or 25×10³ or 1×10⁵ oocysts of the egg-attenuated lines.Oocyst production was measured daily between 6 and 13 days afterinoculation and the results obtained are given in Table 8 below.

                  TABLE 8                                                         ______________________________________                                        Reproduction of E. necatrix (H), (NA 23.sub.7), (NA 29.sub.7), (NA            33.sub.7)                                                                     and (NA 36.sub.7) in chickens                                                              Dose of    Mean number of oocysts                                             ooxysts given                                                                            produced per bird                                     Parasite     (× 10.sup.-3)                                                                      (× 10.sup.-6)                                   ______________________________________                                        E. necatrix (H).sup.+                                                                      5          30.90   25.51                                                                              26.33                                                                              23.73                               E. necatrix (NA 23.sub.7)                                                                  25         1.29                                                               100        1.69*                                                 E. necatrix (NA 29.sub.7)                                                                  25                 1.34                                                       100                3.30                                          E. necatrix (NA 33.sub.7)                                                                  25                      0.56                                                  100                     1.84                                     E. necatrix (NA 36.sub.7)                                                                  25                           0.04                                             100                          0.87                                ______________________________________                                         .sup.+ included for each experiment as an internal standard                   *2 birds died from coccidiosis                                           

Very few oocysts were produced following inoculation with E. necatrix(NA 36₇) even when the infective dose contained 1×10⁵ oocysts. The peakof oocyst production for E. necatrix (NA 36₇) and E. necatrix (H)occurred between 6 and 8 and 10 days after inoculation, respectively.

Immunogenicity of E. necatrix (NA 29₇) in chickens maintained in litterpen isolators

In an experiment similar to that described previously for E. necatrix(NA 22₇) the immunogenicity of E. necatrix (NA 29₇) was determined. Sixgroups of 30, 3-weeks-old chickens kept in litter pen isolators wereused. Two groups of chickens were given a primary infection of 5×10³oocysts of E. necatrix (NA 29₇), two groups a primary infection of 1×10²oocysts of E. necatrix (H) and the remaining two groups were leftuninfected. Subsequently all groups were challenged with a dose of 5×10⁴oocysts per chicken of E. necatrix (H). The body weights of all chickenswere measured at the times of primary infection (day 0), at challenge(day 41) and 7 days after challenge (day 48); all chickens being killedat this latter time and their lesions graded. During the period ofimmunisation, litter in the isolators was sprayed with water 11 timesbetween 8 and 24 days after primary infection to provide conditionsfavourable for the recycling of parasites. The results which wereobtained are given below in Table 9 showing that the dose of 5×10³oocysts provides adequate protection against subsequent challenge withthe parent pathogenic strain. Two of the chickens given a primaryinfection of E. necatrix (H) died as a result of acute coccidiosisbetween 17 and 19 days after inoculation, but there were no deathsassociated with primary infection with E. necatrix (NA 29₇).

Chickens given primary infections of E. necatrix (H) were completelyimmune to subsequent challenge as judged by body weight gain and lesionscore. Some lesions were observed in chickens given E. necatrix (NA 29₇)primary infection, but changes in body weight gain were notsignificantly different from those of the control group. In contrast,unimmunised birds had severe lesions as a result of challenge (two birdsdied) and the weight gain was significantly depressed (P<0.001) comparedwith the control group.

                                      TABLE 9                                     __________________________________________________________________________    Body weight changes of chickens given E. necatrix (H) and (NA29)              and maintained in litter pen isolators                                            Parasite and dose of                                                                        Initial         Challenged with    Mean intestinal          Group                                                                             oocysts given for                                                                           body weight                                                                           Weight gain (g)                                                                       5 × 10.sup.4 oocysts                                                             Weight gain (g)                                                                         lesion score 7 days      No. immunisationψ                                                                           (g) day 0                                                                             day 0 to day 42                                                                       of E. necatrix (H)                                                                     day 42 to day                                                                           after                    __________________________________________________________________________                                                         challenge                1   E. necatrix (NA29) 5 × 10.sup.3                                                       161.1   772.3   YES      144.1     0.4                      2   E. necatrix (NA29) 5 × 10.sup.3                                                       161.1   767.2   NO       175.6                              3   E. necatrix (H) 1 × 10.sup.2                                                          161.3   694.1+  YES      163.4     0.0                      4.  E. necatrix (H) 1 × 10.sup.2                                                          161.7   680.6   NO       165.0                              5.  None          161.4   751.9   YES      6.4***    2.6 (two birds                                                                died)                    6.  None          161.1   678.8φ                                                                            NO       140.1                              __________________________________________________________________________     +Two birds in this group died from infection with  E. necatrix                φA`leak` occurred in this isolator and large numbers of oocysts of E.     acervulina were seen during the regular monitoring of litter up to day 42     ψLitter sprayed with water 11 times during period of immunisation.        ***Significantly different from groups 1, 2, 3, 4 and 6 (P<0.001)        

EXAMPLE 3

An attenuated line of E. acervulina has also been developed by selectionfor precocious development of coccidia grown in chickens. Thisprecocious, "attenuated" line hereinafter referred to as the (HP) lineof E. acervulina may be incorporated with egg attenuated E. necatrix(NA), as described in previous examples, into vaccines for combattingand controlling chicken coccidiosis of poultry, according to a preferredembodiment of the invention.

Development of the precocious, "attenuated" strain E. acervulina (HP)

In a preliminary experiment the prepatent period of the parentpathogenic Houghton (H) strain of E. acervulina was found to be 89 h asdetermined by salt flotations of faecal samples taken at intervals ofone hour.

In order to develop a precocious line, therefore, oocysts were initiallyrecovered 96 h post infection (PI), and thereafter the first oocystsproduced were inoculated into further chickens (LS) and within 2passages the prepatent period had been reduced to 83 h. Some difficultywas experienced at the 4th passage in collecting sufficient oocysts at83 h but, subsequently, it proved possible to reduce the time requiredto collect workable numbers of oocysts to 72 h. At this latter stage itwas necessary to relax the selection pressure for precocious developmentafter 2 unsuccessful attempts to reduce further the collection time, andoocysts were collected after 90 h. The earlist time after infection atwhich it was possible to collect oocysts was 70 h, at the 14th passage.

Parallel chicken passages of the parent pathogenic strain were carriedout, oocysts being collected 120 h PI. The prepatent period after 15passages of the parent (H) strain was found to be 89 h.

Characteristics of the precocious line

1. Reproduction

The reproduction of the HP line was examined after the 12th passage (10passages followed by 2 passages of relaxed selection pressure). Fourgroups of 3-weeks-old LS chickens were inoculated with doses of 1×10² or1×10⁴ oocysts per bird of either E. acervulina (H), or E. acervulina(HP), and the oocyst production of each group during the period from 3to 9 days after inoculation was determined. The results obtained aregiven below in Table 10.

                  TABLE 10                                                        ______________________________________                                        The total mean oocyst output over 9 days per LS chicken                       given either 1 × 10.sup.2 or 1 × 10.sup.4 oocysts                 of E. acervulina (H) or (HP)                                                  Strain and dose of                                                                           Mean oocyst output                                             oocysts given  per bird (× 10.sup.-6)                                   ______________________________________                                        (H)       1 × 10.sup.2                                                                     11.9                                                       (H)       1 × 10.sup.4                                                                     299.5                                                      (HP)      1 × 10.sup.2                                                                     1.7                                                        (HP)      1 × 10.sup.4                                                                     119.0                                                      ______________________________________                                    

Up to the ninth day after infection the parent (H) strain producednumbers of oocysts which were greater than those produced by theprecocious line by factors of 6.9 and 2.5 for inoculations of 1×10² and1×10⁴ oocysts respectively.

2. Histology

Endogenous stages of (HP) line and the (H) strain were studied instained sections of intestine. It was found that development of theendogenous strains of the two parasites was identical up to 60 h PI. At66 h PI, however, gametocytes were found in the (HP) line infection, butdid not appear in the parent (H) strain infection until 80 h. A fewmature 4th generation schizonts were seen in infections with bothparasites at 66 h but subsequently were abundant only in associationwith the (H) strain. It appears, therefore, that gametocytes of the (HP)line develop mainly from 3rd generation schizonts with a small numberdeveloping later from 4th generation schizonts.

Pathogenicity and Immunogenicity

1. Pathogenicity

As in previous examples for E. necatrix, the pathogenicity of E.acervulina (H) and (HP) was measured by comparing the weight gains ofuninfected and infected birds. Birds were weighed individually each dayfrom the day before infection until the 14th day after infection and themean weight gains were calculated. E. acervulina (HP) oocysts used inthis experiment were obtained after the 13th passage (10 passagesfollowed by 3 passages of relaxed selection pressure). Five groups of3-weeks-old weight-matched LS chickens (each group divided into 3sub-groups of 7) were used; four groups being given 1×10⁵ or 1×10⁶oocysts per chicken of either the (H) strain or the (HP) line; and theremaining group being kept as an uninfected control group. The resultsobtained are given diagrammatically in FIG. 3 showing that the weightlosses of birds infected with E. acervulina (HP) were significantly(P<0.001) less than for the (H) strain infected birds. Birds given 1×10⁵oocysts of the (HP) line showed no mean weight loss while birds giventhe same dose of the (H) strain lost weight on day 5 after challenge.The higher dose (10⁶ oocysts) of the (H) strain and (HP) line bothproduced weight loss, though chickens given the (HP) line recoveredsignificantly faster. Four chickens (19%) given the higher dose of the(H) strain died, whilst no birds given the oocysts of the (HP) linedied.

2. Immunogenicity

Oocysts of the (HP) line from the 12th passage (10 passages followed by2 passages of relaxed selection pressure) were used for this experiment.Four groups of 3-weeks-old LS birds (each group made up of 3 subgroupsof 4 birds) were given 1×10⁵ oocysts per bird of the (H) strain or (HP)line and challenged 14 days later with 1×10³ oocysts per bird of eitherparasite. At the time of challenge 2 control groups were also given1×10³ oocysts of either parasite. The results obtained are given belowin Table 11, indicating that chickens given a primary infection of the(HP) line were almost completely immune to challenge with the (H) strainas judged by their oocyst output. These birds were also strongly immuneto subsequent challenge with the (HP) line. These results indicate that,despite a reduction in the reproductive capacity of the (HP) line, it isstill characterised by stages which contain the antigens responsible forprotective immunity.

                  TABLE 11                                                        ______________________________________                                        Cross-immunity between the (H) strain and                                     (HP) line of E.acervulina in LS chickens                                      Immunising   Challenge                                                        parasite     parasite     Oocysts/bird                                        (1 × 10.sup.5 oocysts)                                                               (1 × 10.sup.3 oocysts)                                                               days 4-8 (× 10.sup.-6)                        ______________________________________                                        H            H            0.06                                                HP           HP           0.1                                                 HP           H            6.65                                                --           H            352.9                                               --           HP           56.8                                                ______________________________________                                    

Attenuated, precocious lines of E. acervulina (HP), such as thosedescribed above may be incorporated with the egg-attenuated lines of E.necatrix (NA) of the invention in vaccines for prevention and control ofcoccidiosis in chickens.

I claim:
 1. A process for the production of an attenuated strain of E.necatrix, which comprises:passaging a pathogenic E. necatrix inembryonated eggs for from about 20 to about 60 passages.
 2. A processaccording to claim 1, in which embyronated eggs are inoculated withsporozoites of E. necatrix and oocysts are subsequently removed fromsaid eggs, and in which during the first n egg passages, where n is anumber from 6 to 10, alternate passages are carried out in eggs andchickens.
 3. A process according to claim 1, in which conditions usedduring in ovo culture include the use of a temperature of from 39° to43° C. and turning of the eggs at least once per day.
 4. A processaccording to claim 1, in which the selection pressures compriseselection for precocious development.
 5. A process according to claim 1,in which passaging is carried out in the chorioallantoic membrane ofembryonated eggs; sporozoites of E. necatrix are inoculated into saidmembrane; conditions used during in ovo passaging include the use of atemperature of from 39° to 43° C. and turning of the eggs at least onceper day; during the first n egg passages, where n is a number from 6 to10, alternate egg passages are carried out in eggs and chickens; from 20to 50 egg passages are used; and oocysts are recovered from the eggs. 6.A process according to claim 1, in which from about 30 up to about 50egg passages are used when selection pressures are relaxed duringpassaging.
 7. A process according to claim 1, in which selectionpressures are imposed during passaging and from about 20 up to about 40egg passages are used.
 8. A vaccine for the prevention and control ofcoccidiosis in poultry, which comprises:a live attenuated strain of E.necatrix produced by passaging a pathogenic E. necatrix strain inembryonated eggs for from about 20 to about 60 passages.
 9. A vaccineaccording to claim 8, further comprising food or drinking water.
 10. Amethod for the prevention and control of coccidiosis in poultry, inwhich poultry are vaccinated with a vaccine according to claim
 8. 11. Avaccine according to claim 8, further comprising at least one otherattenuated non-pathogenic strain of Eimeria in addition to E. necatrix.12. A vaccine according to claim 8, further comprising at least oneattenuated, precocious strain of Eimeria.